CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the putative Drosophila ortholog of CLOCK INTERACTING PROTEIN CIRCADIAN

Abstract

The Drosophila circadian clock is driven by a transcriptional feedback loop in which CLOCK-CYCLE (CLK-CYC) binds E-boxes to transcribe genes encoding the PERIOD-TIMELESS (PER-TIM) repressor, which releases CLK-CYC from E-boxes to inhibit transcription. CLOCKWORK ORANGE (CWO) reinforces PER-TIM repression by binding E-boxes to maintain PER-TIM bound CLK-CYC off DNA, but also promotes CLK-CYC transcription through an unknown mechanism. To determine how CWO activates CLK-CYC transcription, we identified CWO target genes that are upregulated in the absence of CWO repression, conserved in mammals, and preferentially expressed in brain pacemaker neurons. Among the genes identified was a putative ortholog of mouse Clock Interacting Protein Circadian (Cipc), which represses CLOCK-BMAL1 transcription. Reducing or eliminating Drosophila Cipc expression shortens period, while overexpressing Cipc lengthens period, which is consistent with previous work showing that Drosophila Cipc represses CLK-CYC transcription in S2 cells. Cipc represses CLK-CYC transcription in vivo, but not uniformly, as per is strongly repressed, tim less so, and vri hardly at all. Long period rhythms in cwo mutant flies are largely rescued when Cipc expression is reduced or eliminated, indicating that increased Cipc expression mediates the period lengthening of cwo mutants. Consistent with this behavioral rescue, eliminating Cipc rescues the decreased CLK-CYC transcription in cwo mutant flies, where per is strongly rescued, tim is moderately rescued, and vri shows little rescue. These results suggest a mechanism for CWO-dependent CLK-CYC activation: CWO inhibition of CIPC repression promotes CLK-CYC transcription. This mechanism may be conserved since cwo and Cipc perform analogous roles in the mammalian circadian clock.

Keywords: ChIP-seq; Drosophila; RNA-seq; activity rhythms; circadian clock; clock gene mutants; feedback loop; transcriptional repression.

Figure 1.. ChIP-seq analysis of CLK and CWO binding sites.

(A) Venn diagram of CWO ChIP-seq targets at ZT2 (yellow) and ZT14 (green). The numbers in the brackets are the total number of targets for each time point, excluding CWO binding sites that map to intergenic regions. The numbers in the circles and the overlap region indicate the numbers of targets present in each category or in both categories, respectively. (B) The top five motifs enriched in CWO binding peaks contain canonical CACGTG E-box sequences. (C) Venn diagram comparing CLK ChIP-seq targets (blue) and CWO ChIP-seq targets (red). The numbers shown are determined as described in panel A. (D) ChIP-seq track showing CLK (green) and CWO-HA (blue) binding sites for the core clock genes tim, vri, per, Pdp1 and cwo at ZT2 and ZT14. Chromatin prepared from flies collected at ZT2 and ZT14, but not IPed, were used as input (gray). Binding peaks are based on the analysis of ChIP-seq data in HOMER (see STAR Methods). See also Figure S1, Tables S1 and S2 and Data S1.

Figure 2.. CWO target genes having prominent CWO binding peaks.

ChIP-seq tracks are shown for CLK (green) and CWO-HA (blue) binding sites for the indicated target genes at ZT2 and ZT 14. Chromatin prepared from flies collected at ZT2 and ZT 14, but not IPed, were used as input (gray). Binding peaks are based on the analysis of ChIP-seq data in HOMER (see STAR Methods). See also Figure S2 and Figure S4.

Figure 3.. Expression of direct CWO targets that are upregulated in cwo⁵⁰⁷³ flies.

RNA-seq analysis was carried out on heads from w¹¹¹⁸ (gray lines) and cwo⁵⁰⁷³ (black lines) flies entrained in LD cycles and collected at the indicated times (see STAR Methods). Graphs show mRNA expression levels of two independent biological replicates (open circles and squares) in fragments per kilobase million (FPKM) for the indicated genes. Asterisks indicate rhythmic expression in w¹¹¹⁸ flies. See also Table S3.

Figure 4.. Levels of per, tim and vri mRNAs in Cipc^Δ64, Cipc overexpression, cwo⁵⁰⁷³ and Cipc^Δ64 cwo⁵⁰⁷³ flies.

Flies were entrained in LD, collected on DD day 1 at the indicated times and mRNA levels were measured from fly heads via quantitative RT-PCR. (A) per mRNA levels were measured in w¹¹¹⁸ (WT, black), Cipc^Δ64 (yellow), tim-Gal4 driven UAS-Cipc (Cipc OE, red), cwo⁵⁰⁷³ (purple) and Cipc^Δ64 cwo⁵⁰⁷³ (green) flies. per mRNA was significantly lower (p<0.03) in Cipc^Δ64 than WT at CT14, significantly lower (p<0.03) in Cipc OE than WT at CT6-CT22, significantly higher (p<0.03) in cwo⁵⁰⁷³ than WT at CT2, significantly lower (p<0.0001) in cwo⁵⁰⁷³ than WT at CT10, significantly higher (p<0.04) in Cipc^Δ64 cwo⁵⁰⁷³ than WT at CT14, significantly higher (p<0.05) in cwo⁵⁰⁷³ than Cipc^Δ64 cwo⁵⁰⁷³ at CT2 and significantly lower (p<0.03) in cwo⁵⁰⁷³ than Cipc^Δ64 cwo⁵⁰⁷³ at CT10 and CT14. (B) tim mRNA levels were measured in the genotypes listed in A. tim mRNA was significantly higher (p<0.01) in Cipc^Δ64 than WT at CT10, significantly lower (p<0.02) in Cipc OE than WT at CT10 and CT18, significantly higher (p<0.03) in cwo⁵⁰⁷³ than WT at CT2, significantly lower (p<0.0001) in cwo⁵⁰⁷³ than WT at CT10 and CT18, significantly lower (p<0.04) in Cipc^Δ64 cwo⁵⁰⁷³ than WT at CT14, significantly higher (p<0.05) in cwo⁵⁰⁷³ than Cipc^Δ64 cwo⁵⁰⁷³ at CT2 and significantly lower (p<0.03) in cwo⁵⁰⁷³ than Cipc^Δ64 cwo⁵⁰⁷³ at CT10. (C) vri mRNA levels were measured in the genotypes listed in A. vri mRNA was significantly higher (p<0.01) in in Cipc^Δ64 than WT at CT10.